Method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense

ABSTRACT

The present invention discloses a method for direct transformation of exogenous DNA into resting spores of  Penicillium amagasakiense . The method includes three steps of culture of  Penicillium amagasakiense  and collection of spores, pretreatment of  Penicillium amagasakiense  spores, and electroporation of  Penicillium amagasakiense  spores by using HDEN method, to obtain  Penicillium amagasakiense  spores with introduction of plasmids to be transformed. In the present invention, non-germinated spores are used as a starting material for introduction of an exogenous molecule, and exogenous DNA is introduced into the resting spores of  Penicillium amagasakiense  by employing the HDEN electrotransformation technique, whereby the complex step of spore germination is omitted, and steps of protoplast preparation or  Agrobacterium -mediated transformation in conventional methods etc. are omitted. Moreover, the transformation efficiency is high, and at least an effect of no less than 6000 positive transformants per transformation reaction system can be achieved.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/CN2017/096981 with a filing date of Aug. 11, 2017, designatingthe United States, now pending, and further claims priority to ChinesePatent Application No. 201610813612.1 with a filing date of Sep. 9,2016. The content of the aforementioned applications, including anyintervening amendments thereto, are incorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to the field of biological technologies,and specifically to a method for direct transformation of exogenous DNAinto resting spores of Penicillium amagasakiense.

2. Description of Related Art

Penicillium amagasakiense is a eukaryotic microorganism and afilamentous fungus. Penicillium amagasakiense is an important strain infermentation industry, with which a variety of important industrialproducts such as glucose oxidase can be produced. Penicilliumamagasakiense is a good host to produce recombinant proteins in geneticengineering. Accordingly, the importance of Penicillium amagasakiense inthe modern fermentation industry is evident.

With molecular genetics as a theoretical basis, and by means of modernmethods of molecular biology, genetic engineering aims at changing theoriginal genetic characteristics of organisms to obtain new varietiesand produce new products by constructing a DNA molecule in vitro withgenes of different sources according to a pre-designed blueprintfollowed by transformation into cells. (For example, an enzyme gene withimportant economic value is transformed into Penicillium amagasakiensecells for high expression of the enzyme protein, thereby producing theenzyme.) In this area, a very important part is introduction of foreignDNA into cells. For different species and different states of the cells,different transformation methods are needed, to enable the foreign DNAto cross the cell wall and the cell membrane, transporting in to thecells.

At present, the main DNA transformation methods for use in the field offilamentous fungi (including Penicillium amagasakiense) includes:

1. Protoplast-mediated transformation: in this method, various enzymesare used to hydrolyze the cell wall of fungal mycelia, to expose thecell membrane, under certain chemical conditions, the cell membrane canabsorb exogenous DNA. However, since the structure and composition ofthe fungal cell wall are complex, and the structures and compositions ofthe cell walls of different species and even different subspecies of thesame species are different, hence the methods of protoplast preparationcannot be unified. Moreover, for many fungi, especially Penicilliumamagasakiense, the protoplast preparation is very difficult, the stepsare extremely cumbersome, and special, expensive cell wall hydrolasesare required.

2. Agrobacterium tumefaciens-mediated transformation: Agrobacteriumtumefaciens is a Gram-negative bacterium that infects plant callus andfilamentous fungi, and able to insert the circular plasmid Ti-DNA whichis stably inherited in cells into the host genome. However, the methodhas cumbersome steps, and is time-consuming; and Agrobacteriumtumefaciens has selectivity to hosts, so this method is not suitable forall the fungi. In addition, the location at which the exogenous DNA isinserted into the host genome is random and unpredictable. The exogenousDNA can only be inserted into the host genome and unable to sustainadrift in the host cytoplasm.

3. Biolistic transformation: in this method, DNA is adsorbed on thesurface of special metal particles, using gunpowder explosion or ahigh-pressure gas acceleration, the metal particles adsorbed with DNAare directly introduced into intact tissues or cells. This methodrequires very expensive special laboratory equipment, and the metalparticles used in the consumables often require to use gold materials.In addition, transformation efficiency of this method is not high, thehost cells have a high death rate under the bombardment of metalparticles, and regeneration is difficult, hence the scope of use islimited.

4. Electroporation: this is a method using short electrical pulse oncells exposed to exogenous DNA, causing exogeneous DNA to enter thecells, thereby changing the genetic characteristics of the cells. Unlikethe Agrobacterium-mediated transformation that requires Agrobacterium asa mediator and the biolistic transformation that requires metalparticles as a mediator, this method does not require a mediator tocarry the exogenous DNA. The traditional electroporation includesexponential decay wave electroporation and square wave electroporation.The energy for exponential decay wave electroporation is particularlyhigh, causes big damage to the cells. Due to microbial cells have simplestructure, presence of cell wall, strong cell resistance, high vitality,and many microbial cells have hard shells, they can withstandhigh-energy electroporation, hence the exponential decay waveelectroporation is generally only for microbial cells. However,mammalian cells (such as human cell lines) are relatively fragile andprone to death compared to microbes. Therefore, the square waveelectroporation is commonly used in mammalian cells. In addition, thesquare wave electroporation is also widely used for in-vivoelectroporation in small animals such as mice.

High-density distributed electrode network (HDEN) electrotransformationtechnique introduced in 2013 is an electrotransformation techniquedeveloped specifically for the characteristics of mammalian cells, withthe purpose of to improve the efficiency of delivery of exogenous DNAand drug to mammalian cells. The technique consists of three parts,first is the form of an electric pulse applied to cell sample, second isthe solution environment where the cell sample is electroporated, andthird is the culturing method of the cell sample and the pre-treatmentmethod before electroporation. Because the technique is developedspecifically for mammalian cells, the above-mentioned three parts aredesigned targeted for the characteristics of mammalian cells.

It is well known in the art that the characteristics and the culturingmethod of microbial cells are vastly different from those of mammaliancells. The structures of mammalian cells and microbial cells aredifferent. Mammalian cells have no cell walls. Microbial cells (such asmost fungi, including Penicillium amagasakiense) have cell walls. Thecell membrane of mammalian cells is also different from the cellmembrane of microbial cells.

Therefore, any technique applied to mammalian cells is difficult to bedirectly applied to microbial cells. Application of the HDEN techniquein mammalian cells such as HEK-293A, Hela, Neuro-2A, MCF-7, C2C12,3T3-L1, CHO, MDCK, HL-60, HUVEC, A375, U251 etc. is reported in currentliteratures. Currently, there is no reports on the use of this techniquein species other than mammalian cells at present. Therefore, whether theHDEN electrotransformation technique can be applied to species otherthan mammalian cells is still unknown.

Filamentous fungi (including Penicillium amagasakiense) are a class ofeukaryotic microorganisms, and the genomes of many types of fungi arepolyploid. The most difficult problem encountered when performinggenetic engineering to a living organism is polyploidy. Becauseengineering on polyploid is often inefficient (for example, genetargeting may hit only one chromosome, and be off target to the otherone or several homologous chromosomes), and during cell division inreproduction of a polyploid, homologous chromosomes are segregated, andthe engineered site is difficult to pass onto all the progenies. This iswell known in the art.

Fungal spores are the major reproductive organs of fungi, and the sporesare dormant and can survive for a long time. Spores are activated andgerminated under appropriate external conditions, to form mycelia forsplit propagation. Most importantly, the spores of many types of fungiare native haploids. Haploid can be genetically engineered directly andthe efficiency is much higher than manipulating polyploids.

However, since the spores are generally dormant, their cell walls arevery thick, and the state of the cell wall and cell membrane of restingspores are different from those of germinated spores and mycelia.Moreover, the intracellular life activity of resting spores is also inthe least exuberant state. Therefore, it is well known in the art thatresting spores have less cell permeability compared to germinated sporesand mycelia. The resting spores hardly exchange substance with theexterior, and is inactive and is generally in dormant state. However,the germinated spores or mycelia, require uptake of nutrients from theexterior for life activities, so permeability of the cell wall and cellmembrane is higher than the resting spores. Therefore, it is verydifficult to introduce exogenous DNA molecules directly into theinterior of sleeping spores.

For the several available well-established techniques, in theprotoplast-mediated transformation, the source of protoplasts in thehost cells, is generally the mycelia body of fungi (polyploid). In theAgrobacterium-mediated transformation, Agrobacterium and fungal sporesare co-cultured on the surface of a solid culture medium for severaldays, exogenous DNA can be introduced into fungal cells, under themediation of Agrobacterium. However, this is not a direct transformationof spores, because fungal spores germinate during co-cultivation andsubsequently Agrobacterium invades the germinated spores to introducethe exogenous DNA. Moreover, in the Agrobacterium-mediatedtransformation, Agrobacterium is needed as a mediator, andtransformation of Agrobacterium has to be carried out before subsequentfungal transformation, hence the operation is very complicated andcumbersome, and the cycle is long.

In the conventional electroporation method (using exponentially decaywave electroporation), the host cells used are germinated fungal spores.For example, in the method developed by Ozeki et al., the spores have tobe germinated first and electroporation can be performed subsequently(specific experimental steps and details for spore germination aredisclosed in the method). This method is a pioneer to typical techniquesin the field, so far there is no significant improvement, and the methodis in use in the field until now. Inventors of the present applicationattempted to transform non-germinated spores by using this method, andit was unsuccessful. So far, there is no successful reports in theliteratures. The method developed by Ozeki et al. is described inliterature 1: OZEKI K, KYOYA F, HIZUME K, KANDA A, HAMACHI M, NUNOKAWAY. Transformation of intact Aspergillus niger by electroporation [J].BiosciBiotechnolBiochem, 1994, 58(12): 2224-2227.

At present, there is no method or any report, that is able to introduceexogenous DNA molecule directly into resting (non-germinated) fungalspores in the absence of a mediator. This is also a technical problemthat has not been overcome in the art.

SUMMARY OF THE INVENTION

In view of the existing defects in the art, an objective of the presentinvention is to provide a method for direct transformation of exogenousDNA into resting spores of Penicillium amagasakiense, in which the stepof fungal spore germination is bypassed, and the exogenous DNA isdirectly introduced into resting spores by using HDENelectrotransformation technique.

To achieve the above objective, the present invention employs thefollowing technical solutions:

A method for direct transformation of exogenous DNA into resting sporesof Penicillium amagasakiense includes the steps of:

1) Culture of Penicillium amagasakiense and Collection of Spores

Penicillium amagasakiense is inoculated onto a surface of a solid agarmedium, and cultured until the Penicillium amagasakiense spores areovergrown on the surface of the medium, the Penicillium amagasakiensespores are washed off from the surface of the medium, suspension of thespores is aspirated off and filtered to remove mycelia, and filtratecontaining the spores is collected, and centrifuged to collect thepelleted resting spores;

2) Pretreatment of Penicillium amagasakiense Spores

the spores are re-suspended in an electroporation buffer, andcentrifuged to collect the spore pellets, the re-suspension andcentrifugation steps are repeated 3-4 times, and the last collectedspore pellets are re-suspended in the electroporation buffer, to obtainan Penicillium amagasakiense spore suspension with a spore concentrationof 10⁴-10¹¹ spores/ml,

in which the electroporation buffer consists of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) having a final concentration of0.01-100 mmol/L and mannitol having a final concentration of 0.5-5000mmol/L, and the pH of the electroporation buffer is 3.0-9.5; and

3) Electroporation of Penicillium amagasakiense Spores by Using HDENMethod

the Penicillium amagasakiense spore suspension prepared in the abovesteps and a plasmid to be transformed are added to wells of a cellculture plate and mixed uniformly, to obtain a mixture of the spores andthe plasmid, the cell culture plate is placed on an ice bath for 10-15min, electroporation is carried out subsequently by using the HDENmethod using an Etta Biotech X-Porator H1 electroporator, by insertingan electroporator head fitted with a matrix electrode into the mixtureof the spores and the plasmid, and energizing, to generate an electricfield inside the mixture of the spores and the plasmid, the cell cultureplate is placed on the ice bath again for 10-15 min afterelectroporation, and subsequently the mixture of the spores and theplasmid is aspirated off, to obtain resting spores of Penicilliumamagasakiense with introduction of exogenous DNA,

in which ratio of the Penicillium amagasakiense suspension to theplasmid to be transformed is 6-600000 μl of Penicillium amagasakiensespore suspension to 0.1-10000 μg of plasmid to be transformed; and

parameters for the electroporation include: a voltage of 1-6000 V, pulseduration of 2-2000000 ms, and repeat for 1-100 times at an interval of5-50000 ms.

Further, the medium in the step 1) is PDA medium, YPD medium orCzapek-Dox medium, and the preferable PDA medium is with the bestoutcome and the spore production capability is the fastest and thelargest.

In the step 1), Penicillium amagasakiense is at a temperature of 16-40°C. with 15-85% humidity for 3-15 days.

Preferably, in the step 1), Penicillium amagasakiense is cultured at atemperature of 25° C. with 50-60% humidity for 8 days.

Further, in the step 2), the electroporation buffer consists of HEPEShaving a final concentration of 1-10 mmol/L and mannitol having a finalconcentration of 50-100 mmol/L, and the pH of the electroporation bufferwas 5.0-7.0.

Preferably, in the step 2), the electroporation buffer consists of HEPEShaving a final concentration of 1 mmol/L and mannitol having a finalconcentration of 50 mmol/L, and the pH of the electroporation buffer was7.0.

In the step 2), the Penicillium amagasakiense spore suspension isobserved under a microscope before electroporation, to confirm that thespore suspension is free of contamination with mycelia and the sporesare non-germinated, and subsequently electroporation is carried out.

Further, in the step 3), the plasmid to be transformed is recombinantplasmid AnEp8-hygro, and the recombinant plasmid AnEp8-hygro isconstructed with a hygromycin B resistance gene and an AnEp8 plasmid,and the electroporation is carried out by using the Etta BiotechX-Porator H1 electroporator.

Preferably, the ratio of the Penicillium amagasakiense suspension to therecombinant plasmid AnEp8-hygro is 60 μl of Penicillium amagasakiensespore suspension to 1 μg of recombinant plasmid AnEp8-hygro.

Further, in the step 3), the voltage is 300-3000 V, the pulse durationis 200-1000000 ms, repeat for 1-50 times at an interval of 500-5000 ms.Preferably, the voltage is 450 V, the pulse duration is 2500 ms, repeatfor 3 times at an interval of 400 ms.

Further, to confirm that the exogenous DNA has been introduced intoresting spores of Penicillium amagasakiense, the step 3) furtherincludes, after electroporating and further standing on an ice bath,aspirating the mixture of the spores and the plasmid off, coating themixture onto a plate containing YPD solid agar medium with a finalconcentration of hygromycin B of 600 μg/ml, culturing the mixture at16-40° C. and preferably 28° C. with 15-85% humidity and preferably50-60% humidity until single colonies are formed, and counting thecolonies.

The hygromycin B (CAS number: 31282-04-9) is dissolved in sterilizedhigh-purity water, to prepare a stock of high concentration, which isdiluted in medium according to a desired ratio before use.

At the same time when the above experimental steps are performed, acontrol group needs to be prepared. The same mixture of “the spores andthe plasmid” that is not electroporated is coated onto another platecontaining YPD solid agar medium with a final concentration ofhygromycin B of 600 μg/ml, and cultured under the same conditions. Nosingle colony is formed in the control group, and the single colonies inthe experimental group are determined to be positive clones.

When single colonies are formed in the experimental group, DNA isextracted from the single colonies in the experimental group, and theexogenous hygromycin B resistance gene which may contained in the DNA isamplified by PCR. The band size of the amplified product is determinedby agarose gel electrophoresis. When the band size is as expected, theamplified product is subjected to Sanger DNA sequencing to determine ifits DNA sequence is consistent with that of the exogenous hygromycin Bresistance gene. If consistent, it can be accurately determined that thepositive clones are successful transformants.

The Penicillium amagasakiense mentioned in the present invention isPenicillium amagasakiense CICC 40341, and the electroporation is carriedout by using the Etta Biotech X-Porator H1 electroporator purchased fromSuzhou Etta Biotech Co., Ltd.

In the present invention, the experimental operations of collection ofnon-germinated spores and the subsequent electroporation process are allconducted in the laboratory at a constant temperature not higher than 23degrees Celsius; the centrifugation steps are all 4° C. coldcentrifugation; and various kinds of liquid exposed to thenon-germinating spores are all pre-cooled on ice in advance, unlessotherwise indicated. The non-germinated spores are prohibited fromcontacting any factors and substances which are able to promote theirgermination (such as YEPD, etc. as a representative medium forgermination), to ensure the dormancy of spores.

During the culture process of Penicillium amagasakiense in the presentinvention, when Penicillium amagasakiense spores are overgrown on thesurface, sterilized water was poured onto the surface of the medium, towash the Penicillium amagasakiense spores off from the surface of themedium. The spore suspension is aspirated with a pipette and filteredusing sterilized lens paper (or fritted glass filter, filter paper,etc.) to remove the mycelia and retain the spores. If there is nofiltration step, the spore suspension will be mixed up with mycelia, andit is unable to determine whether the transformants obtained in thesubsequent steps are positive clones formed after transformation of DNAinto the spores, or false positive clones formed after thetransformation of DNA into the mycelia. The resulting spores aresubjected to chromosome staining, to observe and confirm that thechromosomes in the spores are haploid.

The water used to prepare the solid agar medium in the present inventionshould be MillQ-grade high-purity water or double distilled water havinga resistivity of not lower than 18.2 MΩ-cm used in molecular biology.

When Penicillium amagasakiense spores are electroporated by using theHDEN method, the Penicillium amagasakiense spore suspension and theplasmid to be transformed are added at a proper ratio to the wells of acell culture plate and mixed evenly. The cell culture plate was placedon an ice bath. For example, in a well of a 96-well cell culture plate(Nunclon Surface 96-well cell culture plate from NUNC, Cat. No. 167008),60 μl of the Penicillium amagasakiense spore suspension and 1 μg of therecombinant plasmid AnEp8-hygro are added. The cell culture plate canalso be a 384-well plate, a 24-well plate, a 6-well plate, or otherlarger or smaller containers, and subsequently the mixture system of thespore and the plasmid can be enlarged or reduced according to the volumesize ratio of the containers.

In the present invention, the above technical solution is employed. Itis very simple to use non-germinated spores as a starting material forintroducing exogenous molecules because the complicated step ofgermination of spores can be omitted. More importantly, polyploidy mighthave occurred in the germinated spores, whereas non-germinated sporesare guaranteed to be haploid. In many applications of geneticengineering, the host cells have to be haploid in order to achieve thedesired result or efficiency. Meanwhile, the HDEN electrotransformationtechnique is utilized in the present invention to introduce exogenousDNA into resting spores of Penicillium amagasakiense. In the HDENelectrotransformation technique, a high-density matrix electrode thatproduces a highly uniform and intense electric field is used. Each cellwithin the electric field receives an electroporation condition which isalmost exactly the same. During operation, the cells are placed in acommon container, such as a cell culture plate, and followed byinserting an electroporator head with a matrix electrode into thecontainer, and energized. In the traditional electroporation technique,a special electroporation cuvette is generally used, cells are placedbetween two metal plates positioned in parallel and subsequently themetal plates are electrified to form an electric field forelectroporation. Therefore, the discharge modes in the two methods arecompletely different. In the former one, the electrode is inserted intothe cell suspension and an electric field is generated within, whereasin the latter one, an electric field is generated outside of the wholecell suspension. In the former one, the electrode head is formed by manymetal pins, and a voltage is generated between the pins, whereas in thelatter one, only two metal plates are present, i.e. a positive electrodeplate and a negative electrode plate, and a voltage is generated betweenthe two plates. The HDEN technique also basically eliminates the cathodeeffect existing in the traditional electroporation technology, to avoidthe generation of large amounts of hydroxide ions, so as to avoidkilling the cells and improve the cell survival rate afterelectroporation. However, the cathode effect is difficult to beeliminated in the traditional electroporation technology. Further, intraditional electroporation methods, the electroporation is performedonly once. This is because if multiple electroporations are performed,the cell death rate will be increased greatly. However, in the HDENmethod of the present invention, multiple electroporations may beperformed, and the effects of the multiple electroporations can besuperimposed without significantly increasing the cell death rate.

The HDEN electrotransformation technique is an electrotransformationtechnique developed specifically for the characteristics of mammaliancells, to improve the efficiency of delivery of exogenous DNA and drugto mammalian cells. It is well known in the art that the characteristicsand the culturing methods of microbial cells are vastly different fromthose of mammalian cells. The structures of mammalian cells andmicrobial cells are different. Mammalian cells have no cell walls.Microbial cells (such as most fungi, including Penicilliumamagasakiense) have cell walls. The cell membrane mammalian of cells isalso different from the cell membrane of microbial cells. Even in thesame organism, the structures, states, and chemical compositions ofrespective cell membrane and cell wall of different tissues, differentorgans or different cell types are also different. Even the structures,states, and chemical compositions of respective cell membrane and cellwall of the same tissue, the same organ, and the same cell type of thesame organism are also different in different stages of growth anddevelopment or in different external environments. The cell wall andcell membrane are barriers that block exogenous molecules to enter thecell. Depending on different barriers (with different structures anddifferent chemical compositions), the methods to break through thebarriers are different. In addition, different exogenous molecules whenencountering the same barrier, because of their different structures,molecular weights, volumes and chemical compositions, the methods bywhich these different exogenous molecules break through the same barrierare also different. If encounter with different barriers, the methodsand mechanisms by which different exogenous molecules break through thedifferent barriers are even vastly different.

Therefore, any technique applied to mammalian cells is difficult to beapplied directly to microbial cells. There are no reports of the use ofHDEN technique in species other than mammalian cells at present. In thepresent invention, the exogenous DNA is introduced into resting sporesof Penicillium amagasakiense by using the HDEN technique according tothe characteristics of Penicillium amagasakiense cells. In thisinvention, the culture method of the cell sample, the pre-treatmentmethod before electroporation, the solution environment of the cellsample during the electroporation, and the form of an electric pulseapplied on the cell sample are determined. Employing the method of thepresent invention is very simple and rapid, in which the wild spores canbe directly used as a raw material, preparing the spores in thecompetent state is not required, and removing the cell wall of thespores by various cumbersome methods (e.g. enzymatic hydrolysis) is notrequired. The method just requires washing the wild spores andre-suspending them in the electroporation buffer, in which the steps arevery simple. In the conventional protoplast-mediated transformation, aspecial cell wall hydrolase, and complex steps of preparing andregenerating protoplast are required. In the conventionalelectroporation, spores required germination treatment prior to use,electroporation is performed after the spores are germinated, and thetransformation efficiency is low. In the conventionalAgrobacterium-mediated transformation, Agrobacterium is transformedfirst and followed by subsequent transformation of Penicilliumamagasakiense. In the conventional biolistic transformation, it requiresexpensive consumables, and the transformation efficiency is low.

The transformation efficiency of the method in the present invention ishigh, and at least an effect of no less than 6000 positive transformantsper transformation reaction system is achieved. For example, the size ofthe AnEp8-hygro plasmid used in Example 1 is 12.4 kb. Using 1 μg ofplasmid (about 124.26 fmol plasmid molecules), electroporation of 6×10⁶non-germinated resting spores of Penicillium amagasakiense results in noless than 8000 positive transformants.

It is well known that the plasmid size is inversely proportional to thetransformation efficiency. The larger the plasmid size, the moredifficult to penetrate cross the cell wall and cell membrane. The 12.4kb plasmid used in the present invention is a very large plasmid. It isreported in the literature that the size of the plasmids used in most ofthe transformation of Penicillium amagasakiense (protoplast-mediatedtransformation, and electroporation) is less than 8 kb.

Currently there are various methods describing on transformationefficiency, including the number of positive transformants produced by agiven μg of plasmids; the number of positive transformants produced by agiven number of plasmids (number of moles, number of DNA molecules); andthe number of positive transformants produced by a given number of hostcells, etc. In the present invention, various indicators of number ofhost cells, plasmid quality, plasmid number and plasmid molecular weightetc. are disclosed. The transformation efficiency of a transformationsystem evaluated in any way can be calculated and converted by means ofthe indicators disclosed in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an agarose gel electrophoresis to confirm whether a samplecontains the hygromycin resistance gene after PCR amplification inExample 1.

DESCRIPTION OF THE EMBODIMENTS

The following examples are provided for a better understanding of thepresent invention; however, the present invention is not limitedthereto.

All the experimental process should follow the principle of asepticcondition while conducting the microbial experiments, and theinstruments, consumable materials, and reagents should be sterilized.

Example 1

A method for direct transformation of exogenous DNA into resting sporesof Penicillium amagasakiense includes the following steps:

1) Culture of Penicillium amagasakiense and Collection of Spores

In a 15-cm Petri dish, a solid agar medium (PDA medium) was prepared.Penicillium amagasakiense CICC 40341 was inoculated onto the surface ofthe solid agar medium and cultured at a temperature of 25° C. with50-60% humidity for 8 days, to allow Penicillium amagasakiense spores toovergrow on the surface of the medium.

Sterilized water was poured onto the surface of the medium, to wash downthe Penicillium amagasakiense spores off from the surface of the medium(by vibrating, or gently scratching with a sterilized smooth glassspreading rod). The spore suspension was aspirated with a pipette andfiltered using sterilized lens paper (or fritted glass filter, filterpaper, etc.) to remove the mycelia and retain the spores. The filteredliquid was placed in a centrifuge tube and centrifuged to collect thepelleted resting spores, and the supernatant was discarded. Thecollected spores were subjected to chromosome staining, to observe andconfirm that the chromosomes in the spores are haploid.

2) Pretreatment of Penicillium amagasakiense Spores

The spore pellets were re-suspended in an electroporation buffer (wherethe volume of the electroporation buffer added should fill up thecentrifuge tube), centrifuged again to collect the spore pellets, andthe supernatant was discarded. After repeating the above steps twice,the spores were re-suspended in the electroporation buffer again, andobserved under a microscope, to confirm that the spore suspension wasfree of contamination with mycelia and the spores were non-germinated.When the spores were finally re-suspended in the electroporation buffer,the volume of the electroporation buffer was controlled to maintain aspore concentration in the Penicillium amagasakiense spore suspension of10⁸ spores/ml.

The electroporation buffer consisted of HEPES having a finalconcentration of 1 mmol/L and mannitol having a final concentration of50 mmol/L, and the pH of the electroporation buffer was 7.0.

3) Electroporation of the Penicillium amagasakiense Spores by Using HDENMethod

60 μl of the Penicillium amagasakiense spore suspension and 1 μg ofrecombinant plasmid AnEp8-hygro were added to one well of a 96-well cellculture plate and mixed evenly, to obtain a mixture of the spores andthe plasmid. The cell culture plate was placed on an ice bath for 10min, electroporation was carried out subsequently by using the HDENmethod using an Etta Biotech X-Porator H1 electroporator, by insertingan electroporator head fitted with a matrix electrode into the mixtureof the spores and the plasmid, and energizing, to generate an electricfield inside the mixture of the spores and the plasmid. The cell cultureplate was placed on the ice bath again for 10 min after electroporation,and subsequently the mixture of the spores and the plasmid was aspiratedoff, to obtain resting spores of Penicillium amagasakiense withintroduction of exogenous DNA.

In this example, the parameters for electroporation include: a voltageof 450 V, pulse duration of 2500 ms, and repeat for 3 times at aninterval of 400 ms.

4) Confirmatory Experiment

The aspirated mixture of the spores and the plasmid was coated onto aplate containing YPD solid agar medium with a final concentration ofhygromycin B of 600 μg/ml, and cultured at a temperature of 28° C. with50-60% humidity until single colonies were formed. The colonies werecounted and the transformation efficiency was calculated.

At the same time when the above experimental steps were performed, acontrol group was prepared. The same mixture of “the spores and theplasmid” that was not electroporated was coated onto another platecontaining YPD solid agar medium with a final concentration ofhygromycin B of 600 μg/ml, and cultured under the same conditions. Nosingle colony was formed in the control group, and the single coloniesin the experimental group were determined to be positive clones.

When single colonies were formed in the experimental group, DNA wasextracted from the single colonies in the experimental group, and theexogenous hygromycin B resistance gene which may contained in the DNAwas amplified by PCR. The band size of the amplified product wasdetermined by agarose gel electrophoresis. The result of electrophoresisis shown in FIG. 1. The direction of electrophoresis is from bottom totop. The Marker is Takara 250 bp DNA ladder marker, lane 1 is negativecontrol, and a clear band is observed at about 1 kb in lane 2, which isconsistent with the band of hygromycin B resistance gene, indicatingsuccessful transformation. That is to say the lane 2 is the band of theamplified hygromycin B resistance gene. Sanger DNA sequencing resultshows that the DNA sequence is consistent with the exogenous hygromycinB resistance gene, hence it can be accurately determined that thepositive clone in this example is a successful transformant.

The size of the AnEp8-hygro plasmid in this example was 12.4 kb, and 1μg plasmid (about 124.26 fmol plasmid molecules) was used, with which6×10⁶ non-germinated resting spores of Penicillium amagasakiense wereelectroporated, to produce no less than 8000 positive transformants.

The recombinant plasmid AnEp8-hygro in this example was constructed witha hygromycin B resistance gene and an AnEp8 plasmid. The construction ofthe recombinant plasmid AnEp8-hygro was as follows:

The hygromycin B resistance gene is as shown in SEQ ID NO. 1.

The protein sequence encoded is as shown in SEQ ID NO. 2.

The hygromycin B resistance gene was amplified by PCR using primers:

F:  (SEQ ID NO. 4) CATTAGCTAGCATGAAAAAGCCTGAACTCACCG R:  (SEQ ID NO. 5)TCTGGCGCGCCCTATTCCTTTGCCCTCGG

PCR system (50 μL): template 3 μL, primer F (10 μM) 2 μL, primer R (10μM) 2 μL, 2×Taq PCR mix 25 μL, and ddH₂O top up to 50 μL.

PCR program: 94° C. for 10 min, 35 cycles of (94° C. for 30 s, 61.8° C.for 30 s, and 72° C. for 90 s), and 72° C. for 10 min.

The PCR product was confirmed by agarose gel electrophoresis, and aftercorrect detection, the PCR product was recovered by using ThermoGeneJETGel Extraction and DNA Cleanup Micro Kit.

The AnEp8 plasmid was a gift from U.S. Fungal Genetics Stock Center(FGSC), and was described in literature 2: STORMS R, ZHENG Y, LI H,SILLAOTS S, MARTINEZ-PEREZ A, TSANG A. Plasmid vectors for proteinproduction, gene expression and molecular manipulations in Aspergillusniger [J]. Plasmid, 2005, 53(3): 191-204.

The AnEp8 plasmid in the literature 2 was used. The AnEp8 plasmid has asequence as shown in SEQ ID NO.3. The AnEp8 plasmid was extracted usingplasmid extraction kit available from Shanghai Sangon Biotech. The AnEp8plasmid and purified PCR product of hygromycin B resistance gene weredouble-digested by Fastdigest restriction endonucleases NheI and AscIfrom Fermentas, subjected to agarose gel electrophoresis, and extracted(by using ThermoGeneJET Gel Extraction and DNA Cleanup Micro Kit). Thehygromycin B gene was ligated to the AnEp8 plasmid by using T4 DNAligase (product of Fermentas). The enzymatic cleavage and ligationoperations were carried out strictly according to manufacturer'sinstruction. Subsequently, Escherichia coli transformation wasperformed, to construct a recombinant plasmid Anep8-hygro. Therecombinant plasmid Anep8-hygro was subjected to Sanger sequencing andafter confirmed by double-digestion, the Escherichia coli cells weremass cultured, and the recombinant plasmid was extracted by using aplasmid extraction kit (EndoFree Plasmid Maxi Kit) from Qiagen(according to manufacturer's instruction).

Example 2

A method for direct transformation of exogenous DNA into resting sporesof Penicillium amagasakiense includes the following steps:

1) Culture of Penicillium amagasakiense and Collection of Spores

In a 15-cm Petri dish, a solid agar medium (YPD medium) was prepared.Penicillium amagasakiense CICC 40341 was inoculated onto the surface ofthe solid agar medium and cultured at a temperature of 16° C. with15-50% humidity for 15 days, to allow Penicillium amagasakiense sporesto overgrow on the surface of the medium.

Sterilized water was poured onto the surface of the medium, to wash downthe Penicillium amagasakiense spores off from the surface of the medium(by vibrating, or gently scratching with a sterilized smooth glassspreading rod). The spore suspension was aspirated with a pipette andfiltered using sterilized lens paper (or fritted glass filter, filterpaper, etc.) to remove the mycelia and retain the spores. The filteredliquid was placed in a centrifuge tube and centrifuged to collect thepelleted resting spores, and the supernatant was discarded. Thecollected spores were subjected to chromosome staining, to observe andconfirm that the chromosomes in the spores are haploid.

2) Pretreatment of Penicillium amagasakiense Spores

The spore pellets were re-suspended in an electroporation buffer (wherethe volume of the electroporation buffer added should fill up thecentrifuge tube), centrifuged again to collect the spore pellets, andthe supernatant was discarded. After repeating the above steps twice,the spores were re-suspended in the electroporation buffer again, andobserved under a microscope, to confirm that the spore suspension wasfree of contamination with mycelia and the spores were non-germinated.When the spores were finally re-suspended in the electroporation buffer,the volume of the electroporation buffer was controlled to maintain aspore concentration in the Penicillium amagasakiense spore suspension of10¹¹ spores/ml.

The electroporation buffer consisted of HEPES having a finalconcentration of 0.01 mmol/L and mannitol having a final concentrationof 0.5 mmol/L, and the pH of the electroporation buffer was 3.0.

3) Electroporation of the Penicillium amagasakiense Spores by Using HDENMethod

6 μl of the Penicillium amagasakiense spore suspension and 0.1 μg ofrecombinant plasmid AnEp8-hygro were added to one well of a 96-well cellculture plate and mixed evenly, to obtain a mixture of the spores andthe plasmid. The cell culture plate was placed on an ice bath for 15min, electroporation was carried out subsequently by using the HDENmethod using an Etta Biotech X-Porator H1 electroporator, by insertingan electroporator head fitted with a matrix electrode into the mixtureof the spores and the plasmid, and energizing, to generate an electricfield inside the mixture of the spores and the plasmid. The cell cultureplate was placed on the ice bath again for 15 min after electroporation,and subsequently the mixture of the spores and the plasmid was aspiratedoff, to obtain resting spores of Penicillium amagasakiense withintroduction of exogenous DNA.

In this example, the parameters for electroporation include: a voltageof iv, pulse duration of 2000000 ms, and repeat for 100 times at aninterval of 5 ms.

4) Confirmatory Experiment

The aspirated mixture of the spores and the plasmid was coated onto aplate containing YPD solid agar medium with a final concentration ofhygromycin B of 600 μg/ml, and cultured at a temperature of 16° C. with15-50% humidity until single colonies were formed. The colonies werecounted and the transformation efficiency was calculated.

At the same time when the above experimental steps were performed, acontrol group was prepared (as described in Example 1).

When single colonies were formed in the experimental group, DNA wasextracted from the single colonies in the experimental group, and thepositive clone in this example was confirmed as a successfultransformant by the method as described in Example 1.

The size of the AnEp8-hygro plasmid in this example was 12.4 kb, and 0.1μg plasmid (about 12.426 fmol plasmid molecules) was used, with which6×10⁸ non-germinated resting spores of Penicillium amagasakiense wereelectroporated, to produce no less than 6000 positive transformants.

The construction method of the recombinant plasmid AnEp8-hygro in thisexample was the same as that in Example 1.

Example 3

A method for direct transformation of exogenous DNA into resting sporesof Penicillium amagasakiense includes the following steps:

1) Culture of Penicillium amagasakiense and Collection of Spores

In a 15-cm Petri dish, a solid agar medium (PDA medium) was prepared.Penicillium amagasakiense CICC 40341 was inoculated onto the surface ofthe solid agar medium and cultured at a temperature of 40° C. with60-85% humidity for 3 days, to allow Penicillium amagasakiense spores toovergrow on the surface of the medium.

Sterilized water was poured onto the surface of the medium, to wash downthe Penicillium amagasakiense spores off from the surface of the medium(by vibrating, or gently scratching with a sterilized smooth glassspreading rod). The spore suspension was aspirated with a pipette andfiltered using sterilized lens paper (or fritted glass filter, filterpaper, etc.) to remove the mycelia and retain the spores. The filteredliquid was placed in a centrifuge tube and centrifuged to collect thepelleted resting spores, and the supernatant was discarded. Thecollected spores were subjected to chromosome staining, to observe andconfirm that the chromosomes in the spores are haploid.

2) Pre-Treatment of Penicillium amagasakiense Spores

The spore pellets were re-suspended in an electroporation buffer (wherethe volume of the electroporation buffer added should fill up thecentrifuge tube), centrifuged again to collect the spore pellets, andthe supernatant was discarded. After repeating the above steps twice,the spores were re-suspended in the electroporation buffer again, andobserved under a microscope, to confirm that the spore suspension wasfree of contamination with mycelia and the spores were non-germinated.When the spores were finally re-suspended in the electroporation buffer,the volume of the electroporation buffer was controlled to maintain aspore concentration in the Penicillium amagasakiense spore suspension of10⁴ spores/ml.

The electroporation buffer consisted of HEPES having a finalconcentration of 100 mmol/L and mannitol having a final concentration of5000 mmol/L, and the pH of the electroporation buffer was 7.0.

3) Electroporation of the Penicillium amagasakiense Spores by Using HDENMethod

600000 μl of the Penicillium amagasakiense spore suspension and 10000 μgof recombinant plasmid AnEp8-hygro were added to one well of a 96-wellcell culture plate and mixed evenly, to obtain a mixture of the sporesand the plasmid. The cell culture plate was placed on an ice bath for 10min, electroporation was carried out subsequently by using the HDENmethod using an Etta Biotech X-Porator H1 electroporator, by insertingan electroporator head fitted with a matrix electrode into the mixtureof the spores and the plasmid, and energizing, to generate an electricfield inside the mixture of the spores and the plasmid. The cell cultureplate was placed on the ice bath again for 10 min after electroporation,and subsequently the mixture of the spores and the plasmid was aspiratedoff, to obtain resting spores of Penicillium amagasakiense withintroduction of exogenous DNA.

In this example, the parameters for electroporation include: a voltageof 6000V, pulse duration of 2 ms, and repeat for 1 time at an intervalof 50000 ms.

4) Confirmatory Experiment

The aspirated mixture of the spores and the plasmid was coated onto aplate containing YPD solid agar medium with a final concentration ofhygromycin B of 600 μg/ml, and cultured at a temperature of 40° C. with60-85% humidity until single colonies were formed. The colonies werecounted and the transformation efficiency was calculated.

At the same time when the above experimental steps were performed, acontrol group was prepared (as described in Example 1).

When single colonies were formed in the experimental group, DNA wasextracted from the single colonies in the experimental group, and thepositive clone in this example was confirmed as a successfultransformant by the method as described in Example 1.

The size of the AnEp8-hygro plasmid in this example was 12.4 kb, and10000 plasmid (about 1242600 fmol plasmid molecules) was used, withwhich 6×10⁶ non-germinated resting spores of Penicillium amagasakiensewere electroporated, to produce no less than 7000 positivetransformants.

The construction method of the recombinant plasmid AnEp8-hygro in thisexample was the same as that in Example 1.

Example 4

In this example, the parameters for electroporation were the same asthose in the Example 1 except that the voltage was 30 V, the pulseduration was 1000000 ms, electroporated for 50 times atan interval of5000 ms, and no less than 6500 positive transformants were produced.

Example 5

In this example, the parameters for electroporation were the same asthose in Example 1 except that the voltage was 3000 V, the pulseduration was 100 ms, electroporated for 5 times atan interval of 25000ms, and no less than 7200 positive transformants were produced.

The method for introducing exogenous DNA into resting spores ofPenicillium amagasakiense provided in the present invention can be usedto introduce not only the recombinant plasmid AnEp8-hygro, but also anyother plasmids into resting spores of Penicillium amagasakiense.

I claim:
 1. A method for direct transformation of exogenous DNA intoresting spores of Penicillium amagasakiense, characterized in that, themethod comprising steps of: 1) culture of Penicillium amagasakiense andcollection of spores, wherein Penicillium amagasakiense is inoculatedonto a surface of a solid agar medium, and cultured until Penicilliumamagasakiense spores are overgrown on the surface of the medium, thePenicillium amagasakiense spores are washed off from the surface of themedium, suspension of the spores is aspirated off and filtered to removemycelia, and filtrate containing the spores is collected, andcentrifuged to collect the pelleted resting spores; 2) pretreatment ofPenicillium amagasakiense spores, wherein the spores are re-suspended inan electroporation buffer, and centrifuged to collect the spore pellets,the re-suspension and centrifugation steps are repeated 3-4 times, andthe last collected spore pellets are re-suspended in the electroporationbuffer, to obtain an Penicillium amagasakiense spore suspension with aspore concentration of 10⁴-10¹¹ spores/ml, in which the electroporationbuffer consists of 4-hydroxyethyl piperazineethanesulfonic acid having afinal concentration of 0.01-100 mmol/L and mannitol having a finalconcentration of 0.5-5000 mmol/L, and the pH of the electroporationbuffer is 3.0-9.5; and 3) electroporation of Penicillium amagasakiensespores by using HDEN method, wherein the Penicillium amagasakiense sporesuspension prepared in the above steps and a plasmid to be transformedare added to wells of a cell culture plate and mixed uniformly, toobtain a mixture of the spores and the plasmid, the cell culture plateis placed on an ice bath for 10-15 min, subsequently electroporation iscarried out by using the HDEN method using an Etta Biotech X-Porator H1electroporator, by inserting an electroporator head fitted with a matrixelectrode into the mixture of the spores and the plasmid, andenergizing, to generate an electric field inside the mixture of thespores and the plasmid, the cell culture plate is placed on the ice bathagain for 10-15 min after electroporation, and subsequently the mixtureof the spores and the plasmid is aspirated off, to obtain resting sporesof Penicillium amagasakiense with introduction of exogenous DNA, inwhich ratio of the Penicillium amagasakiense suspension to the plasmidto be transformed is 6-600000 μl of Penicillium amagasakiense sporesuspension to 0.1-10000 μg of plasmid to be transformed; and wherein theparameters for the electroporation comprise: a voltage of 1-6000 V,pulse duration of 2-2000000 ms, and repeat for 1-100 times at aninterval of 5-50000 ms.
 2. The method for direct transformation ofexogenous DNA into resting spores of Penicillium amagasakiense accordingto claim 1, characterized in that, the medium in the step 1) is PDAmedium, YPD medium, or Czapek-Dox medium.
 3. The method for directtransformation of exogenous DNA into resting spores of Penicilliumamagasakiense according to claim 1, characterized in that, in thestep 1) Penicillium amagasakiense is cultured at a temperature of 16-40°C. with 15-85% humidity for 3-15 days.
 4. The method for directtransformation of exogenous DNA into resting spores of Penicilliumamagasakiense according to claim 3, characterized in that, in thestep 1) of claim 1, Penicillium amagasakiense is cultured at atemperature of 25° C. with 50-60% humidity for 8 days.
 5. The method fordirect transformation of exogenous DNA into resting spores ofPenicillium amagasakiense according to claim 1, characterized in that,in the step 2), the electroporation buffer consists of 4-hydroxyethylpiperazineethanesulfonic acid having a final concentration of 1 mmol/Land mannitol having a final concentration of 50 mmol/L, and the pH ofthe electroporation buffer is 7.0.
 6. The method for directtransformation of exogenous DNA into resting spores of Penicilliumamagasakiense according to claim 1, characterized in that, thePenicillium amagasakiense spore suspension in the step 2) is observedunder a microscope before electroporation, to confirm that the sporesuspension is free of contamination with mycelia and the spores arenon-germinated, and subsequently electroporation is carried out.
 7. Themethod for direct transformation of exogenous DNA into resting spores ofPenicillium amagasakiense according to claim 1, characterized in that,in the step 3), the plasmid to be transformed is recombinant plasmidAnEp8-hygro, and the recombinant plasmid AnEp8-hygro is constructed witha hygromycin B resistance gene and an AnEp8 plasmid.
 8. The method fordirect transformation of exogenous DNA into resting spores ofPenicillium amagasakiense according to claim 7, characterized in that,in the step 3) of claim 1, the ratio of the Penicillium amagasakiensesuspension to the recombinant plasmid AnEp8-hygro is 60 μl ofPenicillium amagasakiense spore suspension to 1 μg of recombinantplasmid AnEp8-hygro.
 9. The method for direct transformation ofexogenous DNA into resting spores of Penicillium amagasakiense accordingto claim 1, characterized in that, in the step 3), the parameters forthe electroporation comprise: a voltage of 450 V, pulse duration of 2500ms, and repeat for 3 times at an interval of 400 ms.
 10. The method fordirect transformation of exogenous DNA into resting spores ofPenicillium amagasakiense according to claim 1, characterized in that,the step 3) further comprises aspirating the mixture of the spores andthe plasmid off, coating the mixture onto a plate containing YPD solidagar medium with a final concentration of hygromycin B of 600 μg/ml,culturing the mixture at a temperature of 16-40° C. with 15-85% humidityuntil single colonies are formed, and counting the colonies.